CN109608550B - 用作诱发抗原特异性t细胞反应的免疫原性增强剂的融合蛋白 - Google Patents

用作诱发抗原特异性t细胞反应的免疫原性增强剂的融合蛋白 Download PDF

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CN109608550B
CN109608550B CN201811287073.8A CN201811287073A CN109608550B CN 109608550 B CN109608550 B CN 109608550B CN 201811287073 A CN201811287073 A CN 201811287073A CN 109608550 B CN109608550 B CN 109608550B
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CN109608550A (zh
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吴嘉茂
章修纲
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TheVax Genetics Vaccine Co Ltd
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Abstract

本申请公开一种用作增强抗原特异性T细胞反应的免疫原增强剂的融合蛋白。所述融合蛋白包含：(a)抗原呈递细胞(APC)结合域或CD91受体结合域；(b)蛋白转导域；及(c)病原体的抗原，其中，所述APC结合域或CD91受体结合域位于所述融合蛋白的N端，且所述病原体的抗原位于所述蛋白转导域的C端。所述蛋白转导域选自：(i)融合多肽，其包含T细胞致敏信号转导肽、连接子及转位肽；(it)T细胞致敏信号转导肽；及(iii)长度为34‑112个氨基酸残基的转位肽。

Description

用作诱发抗原特异性T细胞反应的免疫原性增强剂的融合 蛋白
本申请为“用作诱发抗原特异性T细胞反应的免疫原性增强剂的融合蛋白”发明专利申请的分案申请，母案为进入国家阶段的PCT国际申请，其中，母案的国家申请号为“201380063896.1”，PCT国际申请日为2013年12月03日，PCT国际申请号为PCT/US2013/072753。
技术领域
本发明主要涉及融合蛋白，并且更具体地涉及增强T细胞介导的免疫反应的融合蛋白。
背景技术
分子生物学已使亚单位疫苗的生产成为可能，其中，免疫原为母蛋白或母复合物的片段或亚单位。所需要的是可诱发T细胞致敏反应又能足够灵活的并入来自许多感染源的毒株的序列的稳定疫苗的开发。
发明内容
一方面，本发明涉及一种融合蛋白，其包含：
(a)抗原呈递细胞(APC)结合域或CD91受体结合域，其位于所述融合蛋白的N端；
(b)蛋白转导域，其位于所述APC结合域或CD91受体结合域的C端，所述蛋白转导域选自：
(i)融合多肽，其包含T细胞致敏信号转导肽、连接子及转位肽，其中：
(1)所述T细胞致敏信号转导肽位于所述融合多肽的N端；
(2)所述连接子包含SEQ ID NO:15，其连接所述T细胞致敏信号转导胜肽与所述转位肽；且
(3)所述转位肽的长度为34-112个氨基酸残基，且包含与SEQ ID NO:3、SEQ IDNO:20或SEQ ID NO:4至少90％相同的氨基酸序列；
(ii)T细胞致敏信号转导肽；及
(iii)长度为34-112个氨基酸残基的转位肽，其包含与SEQ ID NO:3、SEQ ID NO:20或SEQ ID NO:4至少90％相同的氨基酸序列；及
(c)病原体的抗原，其位于所述蛋白转导域的C端；
其中：
所述T细胞致敏信号转导肽的长度为28-53个氨基酸残基，且包含与SEQ ID NO:31至少90％相同的氨基酸序列，其中Xaa8为I或L；Xaa10为V、F或A；Xaa11为M或L；Xaa17为L或I；且
若所述蛋白转导域为所述转位肽(biii)，则所述APC结合域或CD91受体结合域无假单胞菌外毒素A(PE)结合域I的氨基酸序列。
在本发明的一个实施方式中，所述APC结合域或所述CD91受体结合域为包含与选自SEQ ID NO:5、SEQ ID NO:9、SEQ ID NO:6、SEQ ID NO:7及SEQ ID NO:8的序列至少90％相同的氨基酸序列的多肽。或者，所述APC结合域选自受体相关蛋白-1(RAP1)区域III、α-2-巨球蛋白受体相关蛋白(A2M)、HIV-Tat、热休克蛋白(HSP)及假单胞菌外毒素A(PE)结合域I。
在本发明的另一个实施方式中，所述融合蛋白无假单胞菌外毒素A(PE)结合域I的氨基酸序列。
在本发明的另一个实施方式中，所述融合蛋白进一步包含位于所述融合蛋白C端的内质网(ER)滞留序列。
在本发明的另一个实施方式中，所述内质网滞留序列包含氨基酸序列Lys-Asp-Glu-Leu(SEQ ID NO:14)。所述ER滞留序列可包含选自SEQ ID NO:14、SEQ ID NO:16至SEQID NO:19的序列。或者，所述ER滞留序列可由选自SEQ ID NO:16至SEQ ID NO:19的序列组成。
在本发明的另一个实施方式中，若所述抗原含有10个或更多抗原决定簇，则所述融合蛋白的C端无内质网滞留序列。
在本发明的另一个实施方式中，所述蛋白转导域为融合多肽(bi)。
在本发明的另一个实施方式中，所述蛋白转导域为T细胞致敏信号转导肽(bii)。
在本发明的另一个实施方式中，所述融合蛋白在所述蛋白转导域与所述抗原之间进一步包含另一连接子，所述另一连接子包含SEQ ID NO:15。
在本发明的另一个实施方式中，所述蛋白转导域为转位肽(biii)。
在本发明的另一个实施例方式中，所述融合蛋白在所述APC结合域或所述CD91受体结合域与所述转位肽之间进一步包含另一连接子，所述另一连接子包含SEQ ID NO:15。
在本发明的另一个实施方式中，所述蛋白转导域包含SEQ ID NO:30。
在本发明的另一个实施方式中，所述APC结合域包含与选自SEQ ID NO:5、SEQ IDNO:9、SEQ ID NO:6、SEQ ID NO:7及SEQ ID NO:8的序列至少95％相同的氨基酸序列。
在本发明的另一个实施方式中，所述APC结合域或所述CD91受体结合域为包含选自SEQ ID NO:5、SEQ ID NO:9、SEQ ID NO:6、SEQ ID NO:7及SEQ ID NO:8的氨基酸序列的多肽。
在本发明的另一个实施方式中，所述T细胞致敏信号转导肽包含与SEQ ID NO:1或SEQ ID NO:2至少90％相同的氨基酸序列。
在本发明的另一个实施方式中，所述T细胞致敏信号转导肽包含选自SEQ ID NO:1及SEQ ID NO:2的氨基酸序列。
在本发明的另一个实施方式中，所述转位肽包含选自SEQ ID NO:3、SEQ ID NO:20及SEQ ID NO:4的氨基酸序列。
在本发明的另一个实施方式中，所述转位肽的长度为34-61个氨基酸残基。
在本发明的另一个实施方式中，前述融合蛋白的蛋白转导域具有以下特征：(i)所述T细胞致敏信号转导肽包含SEQ ID NO:1或SEQ ID NO:2的氨基酸序列；且(ii)所述转位肽包含与SEQ ID NO:3至少95％相同的氨基酸序列。
所述T细胞致敏信号转导肽显示出诱发识别并结合于T细胞上CD28受体的K1(X)2E3(X)4(X)5Y6P7P8P9Y10氨基酸序列(SEQ ID NO:32)的抗体的特征，其中，(X)2为I或L；(X)4为V、F或A；且(X)5为M或L。
另一方面，本发明涉及一种融合蛋白，其由下列各项所组成：
(a)抗原呈递细胞(APC)结合域或CD91受体结合域，其位于所述融合蛋白的N端；
(b)蛋白转导域，其位于所述APC结合域或CD91受体结合域的C端，所述蛋白转导域选自：
(i)融合多肽，其包含T细胞致敏信号转导肽、连接子及转位肽，其中：
(1)所述T细胞致敏信号转导肽位于所述融合多肽的N端；
(2)所述连接子包含SEQ ID NO:15，其连接所述T细胞致敏信号转导肽与所述转位肽；且
(3)所述转位肽的长度为34-112个氨基酸残基，其包含与SEQ ID NO:3、SEQ IDNO:20或SEQ ID NO:4至少90％相同的氨基酸序列；
(ii)T细胞致敏信号转导肽；及
(iii)34-112个氨基酸残基长度的转位肽，，其包含与SEQ ID NO:3、20或4号至少90％相同的氨基酸序列；及
(c)病原体的抗原，其位于所述蛋白转导域的C端；
其中：
所述T细胞致敏信号转导肽的长度为28-53个氨基酸残基，且包含与SEQ ID NO:31至少90％相同的氨基酸序列，其中，Xaa8为I或L；Xaa10为V、F或A；Xaa11为M或L；Xaa17为L或I；且
若所述蛋白转导域为所述转位肽(biii)，则所述APC结合域或所述CD91受体结合域无假单胞菌外毒素A(PE)结合域I的氨基酸序列。
所述抗原呈递细胞(APC)可以选自树突细胞、巨噬细胞、B细胞及单核细胞。
在本发明的一个实施方式中，所述APC的细胞膜包含CD91受体。
另一方面，本发明涉及一种疫苗组合物，其包含：(a)治疗有效量的前述融合蛋白；及(b)佐剂。
所述佐剂为抗原传送剂或免疫增效剂。在本发明的一个实施方式中，所述疫苗组合物包含抗原传送剂而无免疫增效剂。
此外，在另一方面，本发明涉及一种诱导增强的病原体抗原特异性T细胞反应的方法，其包括：对有此需要的受试者施用包含治疗有效量的前述融合蛋白的疫苗组合物；以及由此诱导增强的病原体抗原特异性T细胞反应。
此外，在另一方面，本发明涉及一种杀死通过疾病细胞的细胞膜上的第I类主要组织相容性复合体(MHC)分子呈递抗原的疾病细胞的方法，其包括：对有此需要的受试者施用疫苗组合物，所述疫苗组合物包含治疗有效量的前述融合蛋白；以及由此杀死通过疾病细胞的细胞膜上的第I类MHC分子呈递抗原的疾病细胞。
在本发明的一个实施方式中，所述疾病细胞为癌细胞。
在又一个方面，本发明涉及一种预防、治疗由病原体所引起的感染和/或将由感染所引起的症状减至最少的方法，其包括：对有此需要的受试者施用疫苗组合物，所述疫苗组合物包含治疗有效量的本发明的融合蛋白；以及由此预防、治疗由病原体所引起的感染和/或将由感染所引起的症状减至最少。本发明还涉及前述融合蛋白或疫苗组合物，其用于诱导增强的病原体抗原特异性T细胞反应，或用于杀死通过疾病细胞的细胞膜上的第I类MHC分子呈递抗原的疾病细胞，或用于预防、治疗由病原体所引起的感染和/或将由感染所引起的症状减至最少。
所述病原体可以是选自人乳头瘤病毒(HPV)、猪繁殖与呼吸综合症病毒(PRRSV)、人免疫缺陷病毒(HIV-1)、流感病毒、登革热病毒、丙型肝炎病毒(HCV)、乙型肝炎病毒(HBV)及猪环状病毒2(PCV2)中的至少一种。
在本发明的一个实施方式中，前述的融合蛋白为用于增强有此需要的受试者的抗原特异性细胞毒性T细胞反应。所述融合蛋白在有此需要的受试者体内，亦可用于增强抗原特异性CD4+ T细胞反应，或用作诱导增强的抗原特异性抗体效价反应的免疫原性增效剂。
由下述结合以下附图的优选实施方式的描述将明显可见这些及其他方面，但是在不偏离本说明书的新概念的实质和范围的情况下可影响其中的变化和修改。
附图显示本发明的一种或多种实施方式，并与书面说明共同用于解释本发明的原理。在可能情况下，整个附图使用的附图标记指实施例的相同或相似要素。
附图说明
图1为载体图谱。
图2为显示融合蛋白的SDS-PAGE分析结果的照片。
图3为载体图谱。
图4为显示本发明的一个实施方式的示意图。
图5A显示免疫接种程序表。
图5B为显示融合蛋白的SDS-PAGE分析结果的照片。
图5C-D分别为显示接种不同融合蛋白或安慰剂的动物组的肿瘤大小曲线及无肿瘤小鼠的百分比。
图6为制备融合蛋白的流程图(左图)，以及显示融合蛋白的SDS-PAGE分析结果的照片(右图)。
图7为显示T细胞致敏融合蛋白作用机理的示意图。
图8显示不同物种的CD28的序列比对。
图9A显示免疫接种程序表。
图9B-C分别显示接种不同融合蛋白或安慰剂的动物组的肿瘤大小曲线及存活率。
图10为显示用于由病原体产生含有DNA插入片段的质粒的含有RAP1的载体的示意图。
图11为显示含有各种病原体的抗原的融合蛋白结构的示意图。
图12A-F为显示各种融合蛋白的SDS-PAGE分析结果的照片。
图13A-B显示动物组、用于免疫动物的疫苗与剂量，以及免疫接种程序表。
图14A-D为显示来自已接种安慰剂或含有E716、E718、HCV核心或HBx抗原的融合蛋白的图13A的动物组的CD3+/CD4+脾细胞及CD3+/CD8+脾细胞的体外(ex vivo)抗原特异性免疫反应的分析结果的表格。
图15A-J显示来自已接种安慰剂或含有PCV2(图15A-B)或PRRSV抗原(图15C-J)的融合蛋白的图13A的动物组的CD3+/CD8+脾细胞及CD3+/CD4+脾细胞的体外抗原特异性免疫反应分析的IFNγ+细胞计数。
具体实施方式
在以下仅意欲作为说明的实施例中更具体地描述本发明，因为对本领域的技术人员来说其中的修改和变化将明显可见。现在，详细描述本发明的各种实施方式。参考附图，整个图中相同标号表示相同部分。当在此用于说明书和接着的整个权利要求时，除非上下文另外载明，否则“一”、“一个”及“该”的意思包括多个引用项目。并且，当在此用于说明书和接着的整个权利要求时，除非上下文另外载明，否则“在…中”的意思包括“在…中”及“在…上”。此外，为读者的便利在说明书中可以使用标题及小标题，其不应当影响本发明的范围。因此，以下将更具体地定义用于本说明书的若干术语。
定义
用于本说明书的术语在本发明的上下文中及使用各术语的特定上下文中通常在本领域中具有它们的通常涵义。以下或在本说明书的其他地方将讨论用以描述本发明的某些术语以向从业者提供关于本发明的描述的额外指导。为便利，某些术语可能例如使用斜体和/或引号来突出显示。突出显示的使用不影响术语的范围与意义；在相同上下文中术语的范围与意义相同，无论是否突出显示。将意识到同一件事能够以不止一种方式表达。因此，可选择的语言和同义词可以用于在此讨论的任一一种或多种术语，并无任何特殊意义强加于是否在此详细说明或讨论术语。提供某些术语的同义词。一种或多种同义词的列举不排除其他同义词的使用。本说明书中任何地方的实施例的使用(包括在此所讨论的术语的实施例)仅供说明之用，而非用于限制本发明或任何示例性术语的范围与意义。同样地，本发明不局限于在本说明书中给出的各种实施方式。
除非另有定义，在此使用的所有技术与科学术语与本领域中关于本发明的普通技术人员中的一个通常所理解的具有相同含义。若有相互冲突之处，则以本说明书及其所提供的定义为解释依据。
如在此使用的，“左右”、“约”或“大约”通常指在给出的数值或范围的20％以内，优选在10％以内，并且更优选在5％以内。在此给出的数量是大约的，指如果没明确规定能够推测术语“左右”、“约”或“大约”。
术语“抗原呈递细胞(APC)或辅助细胞”指呈现与其表面上的主要组织相容性复合体(MHC)复合的外源抗原的细胞。T细胞可使用它们的T细胞受体(TCR)来识别这些复合体。这些细胞加工抗原并且向T细胞呈递它们。专业抗原呈递细胞主要类型：树突细胞(DC)、巨噬细胞、单核细胞及若干B细胞。
术语“抗原呈递细胞(APC)结合域”指能够结合抗原呈递细胞(APC)的区域。所述APC结合域可为多肽，其包含与选自SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8及SEQ ID NO:9的序列至少90％相同的氨基酸序列。APC结合域为识别并结合在APC上的受体的配体。
分化簇91(CD91)为在细胞膜中形成受体且参与受体介导的内吞作用的蛋白质。
术语“蛋白转导域”指具有致敏T细胞并且因此增强抗原特异性T细胞反应，和/或将抗原导向或指向(即，靶向)抗原呈递的第I类主要组织相容性复合体(MHC-I)通路(即，细胞毒性T细胞通路)的功能的多肽或融合多肽。
术语“致敏T细胞”通常指致敏CD8+及CD4+ T细胞，并且因此增强CD8+(CTL)及CD4+T细胞对抗原激发的反应。抗原特异性细胞介导的免疫反应通过在对抗原的反应中定量抗原特异性诱导的γ-干扰素的产生来测量。例如，在无致敏信号(即，无蛋白转导域)的情况下，抗原仅能诱导微弱的细胞介导免疫反应，或根本无法诱导细胞介导的免疫反应，即，CD8+及CD4+ T细胞仅制造出少量抗原特异性γ-干扰素，或未制造出抗原特异性γ-干扰素，但在致敏信号(蛋白转导域)存在的情况下，所述抗原可诱导增强的细胞介导的免疫反应。因此，致敏信号(蛋白转导域)的作用在于敏化宿主体内的CD4+及CD8+ T细胞，以使得当宿主日后被抗原激发时，由于先前CD4+及CD8+ T细胞致敏，所述抗原可可诱导增强的抗原特异性细胞介导的免疫反应。
蛋白转导域可为肽和/或多肽，其选自：
(i)融合多肽，其包含T细胞致敏信号转导肽、连接子及转位肽，其中：
(1)所述T细胞致敏信号转导肽位于所述融合多肽的N端；
(2)所述连接子包含SEQ ID NO:15，其连接所述T细胞致敏信号转导肽与所述转位肽；且
(3)所述转位肽的长度为34-112个氨基酸残基，且包含与SEQ ID NO:3、SEQ IDNO:20或SEQ ID NO:4至少90％相同的氨基酸序列；
(ii)T细胞致敏信号转导肽；及
(iii)长度为34-112个氨基酸残基的转位肽，其包含与SEQ ID NO:3、SEQ ID NO:20或SEQ ID NO:4至少90％相同的氨基酸序列。
蛋白转导域可为“融合多肽”，其中，所述融合多肽包含T细胞致敏信号转导肽、连接子及转位肽。例如，所述融合多肽可为多肽“CD28convPEt”。
术语“CD28conv”指CD28保守区，其为“T细胞致敏信号转导肽”。其为诱发CD28激动剂抗体的抗原决定簇。
术语“PEt”指长度为34-112个氨基酸残基的转位肽。
“CD28conv”与“PEt”之间存在连接子。融合多肽“CD28convPEt”的方向或排列方式至关重要，因为“CD28conv”(或T细胞致敏信号转导肽)必须位于PEt(或转位肽)的上游，即，PEt必须位于“CD28conv”的C端以获得增强的T细胞反应。“CD28convPEt”与反向融合肽PEtCD28conv相比可很大程度上提高对CD28conv具有特异性的IgG效价(称为CD28特异性激动剂抗体)。所述CD28特异性激动剂抗体可致敏CD4+及CD8+ T细胞。方向正确的融合多肽CD28convPEt在CD28conv与PEt区域之间含有连接子(RXRXKR)。所述连接子含有抗原呈递细胞(APC)特异性蛋白酶(组织蛋白酶L)切割位点Lys-Arg(KR)。因此，所述融合蛋白RAP1-CD28conv PEt-抗原-K3可经消化后将形成两个片段：RAP1-CD28conv及PEt-抗原-K3。RAP1-CD28conv片段可在溶酶体中进一步被消化，然后通过MHC II通路向APC细胞表面呈递CD28conv的抗原决定簇，其又诱发产生CD28激动剂抗体的体液免疫反应。因此，CD28激动剂抗体由B细胞产生。所述CD28激动剂抗体可结合T细胞表面上的CD28，并预先活化T细胞(CD4+及CD8+ T细胞)。
“T细胞致敏信号转导肽”的长度为28-53个氨基酸残基，且包含与SEQ ID NO:31至少90％相同的氨基酸序列，其中，Xaa8为I或L；Xaa10为V、F或A；Xaa11为M或L；Xaa17为L或I。
所述T细胞致敏信号转导肽包含关键区K1(I/L)2E3(V/F/A)4(M/L)5Y6P7P8P9Y10(SEQID NO:32)，其中，(X)2为I或L；(X)4为V、F或A；(X)5为M或L。
以下实施例使用对小鼠具有特异性的T细胞致敏信号转导肽(TDIYFCKIEFMYPPPYLDNEKSNGTIIH，SEQ ID NO:31，其中，X8为I，X10为F，X11为M)。
图7为以融合蛋白RAP1-CD28convPEt-E7-K3为例显示T细胞致敏融合蛋白的作用机理的示意图。RAP1-CD28convPEt-E7-K3自N端至C端依序包含：(1)位于N端的全长RAP1的区域III；(2)CD28conv；(3)连接子；(4)来自假单胞菌外毒素A的修饰的转位肽；(5)全长第16型HPV E7蛋白；及(6)位于C端作为ER滞留信号的三段KDEL。HPV16E7蛋白被加工成用融合蛋白免疫的受试者细胞内的抗原决定簇。相较于仅含有抗原的传统疫苗，RAP1-CD28convPEt-E7-K3可诱发较佳的细胞毒性T细胞(CTL)反应。设计RAP1-CD28convPEt-E7-K3蛋白来改善APC(如树突细胞)的摄取率并且增强向蛋白酶体途径的HPV16 E7抗原加工，然后通过MHC I复合体呈递。由疫苗RAP1-CD28convPEt-E7-K3诱发的HPV16 E7蛋白特异性CTL免疫反应的作用机理如图7所示：(a)疫苗结合APC(如树突细胞)表面受体(CD91)，并经由内吞作用而内在化；(b1)RAP1-CD28convPEt-E7-K3因组织蛋白酶L蛋白酶在转位肽PEt前的位点进行消化而经过蛋白水解；(b2)或RAP1-CD28convPEt-E7-K3循环至E.R.，并且因弗林蛋白酶在转位肽PEt前的位点而经过蛋白水解；(b3)与此同时，RAP1-CD28conv由溶酶体蛋白酶消化，然后通过MHC II向细胞表面呈递CD28conv的抗原决定簇，并且诱发CD28激动剂抗体的产生，这能预先活化T细胞；(c)最重要的步骤是通过转位肽(PEt)使PEt-E7-K3从溶酶体跨膜转位进入细胞质区室；(d)PEt-E7-K3经蛋白酶体途径进行消化，然后通过MHC I复合体呈递E7的抗原决定蔟，并诱发对E7特异性细胞介导的免疫反应。
图8显示不同物种的CD28保守区序列比对及其共有序列。共有序列中画线的序列(KIEVMYPPPY；SEQ ID NO:32，其中，X2为I，X4为V，X5为M)为CD28激动剂抗体识别与结合的关键区。该关键区可以K1(I/L)2E3(V/F/A)4(M/L)5Y6P7P8P9Y10表示，其中，仅在此的第四个氨基酸残基为物种特异性的：并且在人类、大鼠、猪、牛、绵羊、狗及马中应为V；在小鼠中应为F；在火鸡中应为V。所述关键区序列可用K1(X)2E3(X)4(X)5Y6P7P8P9Y10(SEQ ID NO:32)表示，其中，(X)2为I或L；(X)4为V、F或A；(X)5为M或L。
PEt可包含与SEQ ID NO:3或SEQ ID NO:20至少90％相同的氨基酸序列。例如，PEt的氨基酸序列可为PE的a.a.280-a.a.313(SEQ ID NO:3)、a.a.268-a.a.313(SEQ ID NO:20)、a.a.253-a.a.313或a.a.253-a.a.364(SEQ ID NO:4)。换言之，PEt的氨基酸序列可含有PE区域II(a.a.253至a.a.364；SEQ ID NO:4)的任一区，只要其包含a.a.280-a.a.313(SEQ ID NO:3)必要的片段即可。
抗原可为病原体蛋白、多肽或肽，其可导致由病原体所引发的疾病的原因，或能够在病原体所感染的宿主体内诱发免疫反应；或为在肿瘤细胞内特异性表达的多肽的肿瘤相关抗原(TAA)。所述抗原可选自病原体或癌症细胞，其包括但不局限于人乳头瘤病毒(HPV)、PRRSV、HIV-1、流感病毒、登革热病毒、丙型肝炎病毒(HCV)、乙型肝炎病毒(HBV)、猪环状病毒2(PCV2)、非小细胞肺癌、乳癌、黑色素瘤、淋巴瘤、结肠癌、肝细胞癌，及它们的结合。例如，HPV E7蛋白(E7)、HCV核心蛋白(HCV核心)、HBVX蛋白(HBx)选作开发疫苗的抗原。所述抗原可为两种以上的抗原(选自一或多种病原体蛋白)融合而成的融合抗原。例如，PRRSVORF6与ORF5的融合抗原，或PRRSV与PCV2病原体的抗原蛋白的融合。
内质网滞留序列的作用在于协助从内吞区室至ER的抗原转位，并将其留在内腔中。内质网滞留序列包含序列Lys Asp Glu Leu(KDEL)或RDEL。ER序列可包含序列KKDLRDELKDEL(SEQ ID NO:16)、KKDELRDELKDEL(SEQ ID NO:17)，KKDELRVELKDEL(SEQ IDNO:18)，或基本上上由序列KKDLRDELKDEL(SEQ ID NO:16)、KKDELRDELKDEL(SEQ ID NO:17)，KKDELRVELKDEL(SEQ ID NO:18)组成，或由序列KKDLRDELKDEL(SEQ ID NO:16)、KKDELRDELKDEL(SEQ ID NO:17)，KKDELRVELKDEL(SEQ ID NO:18)组成。
分子量为39kDa(千道尔顿)的受体相关蛋白(RAP1)为ER驻留蛋白，且为低密度脂蛋白受体相关蛋白的分子伴侣(molecular chaperone)。RAP1对CD91具有高结合亲和度(Kd～3nM)，且由三个功能类似的区域构成。
本发明涉及通过根据本发明的融合蛋白的T细胞介导的免疫反应的诱导和增强的发现。以RAP1-CD28convPEt-E7-K3为例，RAP1-CD28convPEt-E7-K3疫苗的策略主要致力于刺激T细胞的产生与活化，其能识别的表达靶抗原E7的HPV16受感染细胞。通过传递抗原至树突细胞，能产生抗原特异性CD8+ T细胞及CD4+ T细胞。第1型辅助CD4+ T细胞尤能有效刺激并增强细胞毒性CD8+ T细胞的免疫反应。同时，适应性免疫系统的这两个部分具有于身体多个位点杀死HPV16受感染细胞或与HPV16相关的肿瘤细胞而不对正常组织造成显著损伤。
术语“受试者”指人类或非人类动物。
术语“治疗”指对已患有癌症或感染、或有该疾病症状或诱因的有此需要的受试者施用有效量的融合蛋白，藉以治愈，减轻，解除，改善，缓解或预防疾病、其症状或其诱因。基于任一适当诊断方法的结果能够由健康护理专业人士识别该受试者。
术语“有效量”指给予治疗的受试者治疗效果所需的活性化合物的量。如本领域的技术人员所公认的，有效剂量将根据施用的途径、赋形剂的使用以及与其他治疗手段共用的可能性而变化。
缩写：CD 28，分化簇28。
实施例
以下给出根据本发明实施方式的例示性的仪器、设备、方法与它们相关的结果，而不意欲限制本发明的范围。请注意，为读者的便利，标题或子标题可以用于实施例，其不应限制本发明的范围。此外，在此提出或说明某些理论；但是，不论它们正确或错误，它们不应当限制本发明的范围，只要本发明为根据其本身实践，不管任一特定理论或实行方案。
实施例1
表达载体的构建
图1显示融合蛋白RAP1-PE268-313-E7-K3的表达载体，其包含RAP1区域3(SEQ IDNO:5)、转位最小基本肽(PE268-313；SEQ ID NO:20)、抗原E7及内质网滞留序列(K3，或KDEL信号；SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:19)。转位最小基本肽(PE268-313；SEQ ID NO:20)为从a.a.268至a.a.313的PE(SEQ ID NO:10号)多肽序列区获得的。
所述表达载体为利用图10所示的质粒RAP1-K3构建。含有RAP-1区域3与K3的融合基因的质粒RAP1-K3(图10)如下产生：通过PCR法合成编码NdeIRAP1-(EcoRI,XhoI)-K3XhoI的DNA片段，然后利用卡那霉素抗药基因连接到质粒pUC18骨架中获得质粒RAP1-K3(图10)。为产生表达载体RAP1-PE268-313-E7-K3(图1)，将编码PE268-313-E7的DNA片段插入质粒RAP1-K3中(图10)。运用类似方法，通过PCR产生编码各种融合肽的DNA片段(图11)，并将其分别插入质粒RAP1-K3(图10)中以产生各种融合蛋白的表达载体(图12A-F)。图3显示融合蛋白RAP1-CD28convPEt-E7-K3的表达载体(图3)。
CD28convPEt片段含有组织蛋白酶L及弗林蛋白酶的切割位点。图4显示融合蛋白RAP1-CD28convPEt-E7-K3的氨基酸序列图谱来说明CD28convPEt片段的重要性。两箭头分别指示组织蛋白酶L及弗林蛋白酶的切割位点。这两个切割位点不仅可用做连接CD28conv与PEt-E7的连接子，还允许融合蛋白的切割，以便将PEt-E7-K3片段从溶体释放至细胞质中。由各种病原体引起的任何其他目的抗原可取代E7来与CD28convPEt融合，之后再将所述融合产物插入到图10的质粒中，以形成图11所示的各种融合蛋白的表达载体。
PE268-313的序列为：pletftrhrqprgweqleqcgypvqrlvalylaarlswnqvdqvir(SEQ IDNO:20)。CD28convPEt的完整序列如下：tdiyfckiefmypppyldneksngtiihrarykrgweqleqcgypvqrlvalylaarlswnqvdqvirgs(SEQ ID NO:30)，画线的序列代表含有组织蛋白酶L与弗林蛋白酶切割位点的连接子序列。
实施例2
蛋白表达
带有蛋白表达载体的大肠杆菌(E.coli)BL21细胞在37℃下，在含有25μg/ml卡那霉素的Luria Bertani培养基中培养。当培养物到达早期对数生长期(A600＝0.1至0.4)时，加入异丙基-β-D-硫代半乳糖苷(IPTG)，使其最终浓度为0.5至2mM以达诱导的目的。在IPTG诱导4小时后，收集细胞并通过声波震荡来干扰。将含有过表达蛋白的包涵体分离出来，并在8M尿素/TN缓冲液(8M尿素、50mM(Tris)、50mM NaCl，pH 8.0)中溶解。
融合蛋白RAP1-PE268-313-E7-K3的再折叠在4℃下，通过50X体积的TNZ缓冲液(50mMTris、50mM NaCl及0.01mM ZnCl2，pH 8.0)透析过夜来进行。再折叠的蛋白在还原(包含二硫苏糖醇；+DTT)及非还原(不含二硫苏糖醇；-DTT)条件下经过SDS-PAGE分析(图2)。分析结果显示，大部分再折叠的蛋白在非还原条件下为单体，显示所述RAP1融合蛋白容易再折叠且未聚集(图2)。
图6A为显示表达融合蛋白RAP1-CD28PEt-E7-K3(其含有小鼠CD28conv或人类CD28conv)并从E.coli细胞的包涵体中萃取而出的流程图。SDS-PAGE分析显示，融合蛋白已充分再折叠(图6B)。
图11显示以如下描述的类似方法表达的融合蛋白的列表：(1)RAP1-PE268-313-E7-K3；(2)RAP1-CD28convPEt-E7-K3；(3)RAP1-CD28PEt-E718-K3；(4)RAP1-HCV核心-K3；(5)RAP1-CD28conv-HCV核心-K3；(6)RAP1-CD28convPEt-HCV核心-K3；(7)RAP1-HBx；(8)RAP1-HBx-K3；(9)RAP1-CD28conv-HBx；(10)RAP1-CD28conv-HBx-K3；(11)RAP1-CD28convPEt-HBx；(12)RAP1-CD28convPEt-HBx-K3；(13)RAP1-PCV2ORF2-K3；(14)RAP1-PE268-313-PCV2ORF2-K3；(15)RAP1-CD28convPEt-PCV2ORF2-K3；(16)RAP1-PE268-313-DGD-K3；(17)RAP1-PE268-313-M12-K3；(18)RAP1-PE268-313-PQAB-K3；(19)RAP1-PE268-313-RSAB-K3；(20)RAP1-CD28convPEt-DGD-K3；(21)RAP1-CD28convPEt-M12-K3；(22)RAP1-CD28convPEt-PQAB-K3；(23)RAP1-CD28convPEt-RSAB-K3。使用上述相同的方法再折叠融合蛋白。SDS-PAGE分析的结果显示，这些融合蛋白均已充分再折叠，并且因此用于制备疫苗(图12A-F)。
实施例3
RAP1-PE268-313-E7-K3抑制第16型人乳头瘤病毒(HPV)E7蛋白所诱导的肿瘤生长
如上所述表达所述融合蛋白PE407-E7-K3及RAP1-PE268-313-E7-K3，并通过SDS-PAGE检查蛋白再折叠(图5B)。小鼠通过皮下注射用2x103个TC-01细胞(带有第16型HPV E7基因的小鼠肺部上皮细胞系)激发，以诱导第16型HPV癌。所述TC-01细胞激发后十二天，小鼠每周一次连续3周经皮下注射接种安慰剂(磷酸盐缓冲溶液+磷酸铝)、PE407-E7-K3(200μg/剂)或RAP1-PE268-313-E7-K3(200μg/剂)(均以AS04C(GlaxoSmithKline)为佐剂)(图5A)。为细胞毒性T淋巴细胞增强佐剂的AS04C包含MPL(单磷酯脂A，免疫增效剂)及磷酸铝(用于抗原传递的蛋白质吸收剂)。术语“K3”代表氨基酸序列KDELKDELKDEL(SEQ ID NO:19)。各组的肿瘤大小及无肿瘤动物数均加以记录(图5C-D)。以AS04C为佐剂的两种疫苗PE407-E7-K3及RAP1-PE268-313-E7-K3显著抑制肿瘤生长。但是，接种RAP1-PE268-313-E7-K3的小鼠组具有较高比例的无肿瘤小鼠。其显示疫苗RAP1-PE268-313-E7-K3在抑制肿瘤生长方面与PE407-E7-K3一样有效，或更为有效，而在提高无肿瘤动物的比例方面更好。
实施例4
RAP1-CD28convPEt-E7-K3可抑制第16型人乳头瘤病毒(HPV)E7蛋白所诱导的肿瘤生长并提高存活率
检验有或没有免疫增效剂的融合蛋白PE407-E7-K3及RAP1-CD28convPEt-E7-K3对肿瘤大小及存活率的影响。小鼠经皮下注射以较高剂量(3x104)的TC-01细胞激发。激发后七天，小鼠以每周一次连续3周经皮下注射接种有免疫增效剂GPI-0100或蛋白质吸收剂磷酸铝的安慰剂、PE407-E7-K3(100μg/剂)或RAP1-CD28convPEt-E7-K3(100μg/剂)(图9A)。GPI-0100为Th1/CTL刺激佐剂(免疫增效剂)。各组的肿瘤大小及存活率均加以记录(图9B-C)。当与佐剂GPI-0100结合时，PE407-E7-K3及RAP1-CD28convPEt-E7-K3两者抑制肿瘤生长。意外地，发现RAP1-CD28convPEt-E7-K3抑制肿瘤生长的效果不取决于佐剂。当与所述蛋白质吸收剂磷酸铝结合而未与佐剂GPI-0100结合时，RAP1-CD28convPEt-E7-K3仍可以与免疫增效剂GPI-0100结合时的相同效力有效抑制肿瘤生长(图9B，空心三角形对实心反向三角形)。
相比之下，PE407-E7-K3抑制肿瘤生长的效力取决于佐剂。当与蛋白质吸收剂磷酸铝结合时，PE407-E7-K3的效力低于与所述免疫增效剂GPI-0100结合时的效力(图9B，实心正方形对空心圆形)。
另一方面，就存活率而言，施用与所述免疫增效剂GPI-0100或所述蛋白质吸收剂磷酸铝结合的RAP1-CD28convPEt-E7-K3的小鼠具有比接种与GPI-0100或磷酸铝结合的PE407-E7-K3更高的存活率(图9C)。其显示，RAP1-CD28convPEt-E7-K3即使无免疫增效剂GPI-0100，仍可诱发Th1/CTL免疫反应。所述结果亦显示，作为用于提高动物存活率的疫苗，所述融合蛋白RAP1-CD28convPEt-E7-K3优于PE407-E7-K3。
实施例5
免疫原性分析
测试多种疫苗的免疫原性。小鼠分为下列各组：HPV16 E7、HPV 18 E7、HCV核心、HBV HBx、PCV2 ORF2及PRRSV(图13A)，各组再划分为数个亚组，各亚组依每周一次连续3周经皮下注射安慰剂或针对病原体的一种或多种抗原而设计的疫苗(图13B)。针对PRRSV的疫苗除外，各个疫苗由单一融合蛋白及吸收剂磷酸铝组成，其中，所述单一融合蛋白含有病原体的至少一种抗原。所述抗原为病原体的全长蛋白，或为含有病原体的抗原的至少一种抗原决定簇的非全长蛋白，或为两种以上抗原的融合肽，其中，各抗原为选自病原体的不同蛋白。
免疫接种程序、疫苗及剂量在图13A-B中显示。简言之，小鼠每周一次连续3周接种图13A列出的疫苗。所有小鼠均在最后一次免疫7后天处死，并收集其脾脏。脾细胞经分离后，在有10μg/ml的各种病原体重组抗原的6孔培养板(2x107个细胞/2ml/孔)中培养，以在37℃下在1μg/ml GolgiPlug(BD Pharmingen,San Diego,CA)存在下刺激脾细胞16小时。
刺激后的脾细胞以FACScan缓冲液清洗，并且所述细胞表面标记CD8a、CD4及CD3则以藻红蛋白共轭的单克隆大鼠抗小鼠CD8a抗体、AF700共轭的单克隆大鼠抗小鼠CD4抗体及AF647共轭的单克隆大鼠抗小鼠CD3抗体染色。接着再以根据制造商的说明(BDPharmingen)的Cytofix/Cytoperm套组浸透并固定细胞。细胞内的IFN-γ为以AF488共轭的单克隆大鼠抗小鼠IFN-γ染色，以便量测免疫反应及细胞因子水平。使用Gallios流式细胞术及Kaluza分析软件(Beckman Coulter)进行流式细胞术分析。
测试下列PRRSV疫苗的免疫原性：PE407-PRRSV-K3、RAP1-PE268-313-PRRSV-K3或RAP1-CD28convPEt-PRRSV-K3疫苗。各疫苗含有四种不同融合蛋白的混合物，且各融合蛋白含有选自DGD、M12、PQAB及RSAB的不同抗原(图13A)。小鼠的接种及脾细胞的刺激使用与上述类似的方式进行。简言之，所有小鼠在最后一次免疫后7天处死，并收集脾脏。分离脾细胞，并且分别在有10μg/ml的重组DGD、M12、PQAB或RSAB抗原的6孔培养板(2x107个细胞/2ml/孔)中培养，以在37℃下及1μg/ml GolgiPlug(BD Pharmingen,San Diego,CA)存在下刺激脾细胞16小时。
“DGD”(SEQ ID NO:26)的氨基酸序列如下：
Figure BDA0001849300400000171
Figure BDA0001849300400000172
DGD代表PRRSV ORF7 a.a.64-a.a.123(粗体)、连接子(画线)及ORF7 a.a.64-a.a.123(粗体)的融合抗原。
术语“M12”代表PRRSV ORF1b a.a.1046-a.a.1210的抗原。其氨基酸序列(SEQ IDNO:27)如下：NNKECTVAQALGNGDKFRATDKRVVDSLRAICADLEGSSSPLPKVAHNLGFYFSPDLTQFAKLPIELAPHWPVVSTQNNEKWPDRLVASLRPLDKYSRACIGAGYMVGPSVFLGTPGVVSYYLTKFVKGEAQVLPETVFSTGRIEVDCREYLDDREREVAASLPH。
“PQAB”(SEQ ID NO:28)的氨基酸序列如下：GSSLDDFCYDSTAPQKVLLAFSITYASNDSS SHLQLIYNLTLCELNGTDWLANKFDWA。PQAB代表PRRSV美洲株ORF6 a.a.2-a.a.26及ORF5a.a.31-a.a.63(画线)的融合抗原。
RSAB的氨基酸序列为MGSLDDFCNDSTAAQKLVLAFSITYTPIFVAGGSSSTYQYIYNLTICELN GTDWLSNHFDWA(SEQ ID NO:29)。术语“RSAB”代表PRRSV欧洲株ORF6 a.a.2-28及ORF5a.a.31-64(画线)的融合抗原。
实施例6
融合蛋白RAP1-CD28convPEt-E7-K3的RAP1区域3的片段分别由A2M最小(SEQ IDNO:6)、HIV-Tat最小(SEQ ID NO:7)或HSPs最小(SEQ IDNO:8)取代，以产生融合蛋白A2M-CD28convPEt-E7-K3、Tat-CD28convPEt-E7-K3及HSP-CD28convPEt-E7-K3疫苗。使用与上述类似的方法检测由这些疫苗增强的TC-1肿瘤抑制活性及细胞介导的免疫反应。表1显示各融合蛋白的组分的序列编号(SEQ ID NO)。表2显示测试融合蛋白对动物T细胞介导的免疫反应的影响的及其抗原序列。
表1
Figure BDA0001849300400000181
Figure BDA0001849300400000191
Figure BDA0001849300400000201
表2
Figure BDA0001849300400000202
在免疫原性测定中，通过测量脾细胞中CD3+/CD4+/IFNγ+及CD3+/CD8+/IFNγ+T细胞的数量来评估由各疫苗所诱导的抗原特异性细胞介导的免疫反应。结果显示，所述疫苗RAP1-CD28convPEt-抗原-K3可诱导强烈的T细胞反应。图14B显示，由CD28convPEt-E718-K3所诱发的CD3+/CD4+/IFNγ+T细胞数为RAP1-K3所诱发的细胞数的大约50倍，由CD28convPEt-E718-K3所诱发的CD3+/CD8+/IFNγ+ T细胞数则为RAP1-K3所诱发的细胞数的9倍以上。
疫苗RAP1-CD28convPEt-抗原-K3在诱发T细胞介导的免疫原性方面优于PE407-抗原-K3。例如，图14A显示，由CD28convPEt-E716-K3所诱发的CD3+/CD4+/IFNγ+ T细胞数及CD3+/CD8+/IFNγ+ T细胞数分别为PE407-E716-K3所诱发的T细胞数的大约5倍与7倍。其显示，所述疫苗RAP1-CD28convPEt-E7-K3具有比PE407-E7-K3更好的细胞介导的免疫原性。
当选用的抗原包含10个或多于10个抗原决定簇时，包含RAP1区域III、致敏信号CD28conv(但不含转位肽PEt)、抗原及ER滞留信号的融合蛋白便足以诱发强烈的抗原特异性T细胞介导的免疫反应。图14C显示疫苗RAP1-CD28conv-HCV核心-K3诱发CD3+/CD4+/IFNγ+及CD3+/CD8+/IFNγ+T细胞的数量分别为安慰剂组的20倍及7.6倍的T细胞反应。抗原HCV核心含有11个众所周知的MHC I抗原决定簇。
意外地，所述ER滞留信号对于本发明的融合蛋白诱发强烈的细胞介导的免疫原性来说不是必要的。换言之，在ER滞留序列不存在的情况下，本发明的融合蛋白仍可诱发强烈的T细胞反应。图14D显示，由RAP1-CD28convPEt-HBx(其不含ER滞留信号K3)所诱发的CD3+/CD4+/IFNγ+及CD3+/CD8+/IFNγ+ T细胞的数量为安慰剂组的7倍及74倍。
相较之下，美国专利第7378100B2号及第7335361号显示所述ER滞留信号K3对PE相关的融合蛋白(PE407-抗原-K3)诱发T细胞反应来说是必须的。
还发现，包含RAP1区域III、转位肽PE218-313(但不含致敏信号CD28conv)、抗原及ER滞留信号的融合蛋白优于不含RAP1区域III的PE相关的融合蛋白。图15C-J显示，所述疫苗RAP1-PE268-313-PRRSV-K3所诱发的CD3+/CD4+/IFNγ+及CD3+/CD8+/IFNγ+ T细胞数大于所述疫苗PE407-PRRSV-K3所诱发的T细胞数。
已介绍上述的本发明的示例性实施方式仅用于说明和描述的目的，并未意欲成为全面的，或意欲限制本发明至说明的精确形式。依据以上教示可实现本发明的多种修改及变化。选择并描述实施方式与实施例以便解释本发明的原理与它们的实际应用，为了使本领域的其他技术人员能够利用本发明及各种实施方式及适合预期的特定使用的各种修改。在不偏离本发明的实质和范围的情况下，本发明有关的选择性的实施方式将对本领域的这些技术人员明显可见。因此，本发明的范围由所附权利要求来界定，而非由以上说明和在此描述的示例性实施方式来界定。
序列表
<110> 生控基因疫苗股份有限公司
<120> 用作诱发抗原特异性T细胞反应的免疫原性增强剂的融合蛋白
<130> 10021-00001
<150> US/61733879
<151> 2012-12-05
<160> 32
<170> PatentIn version 3.5
<210> 1
<211> 28
<212> PRT
<213> 人工序列
<220>
<223> hCD28 核心
<400> 1
Thr Asp Ile Tyr Phe Cys Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr
1 5 10 15
Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His
20 25
<210> 2
<211> 53
<212> PRT
<213> 人工序列
<220>
<223> hCD28 最大
<400> 2
Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr Phe Tyr Leu Gln
1 5 10 15
Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys Lys Ile Glu Val
20 25 30
Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile
35 40 45
Ile His Val Lys Gly
50
<210> 3
<211> 34
<212> PRT
<213> 人工序列
<220>
<223> PEt 核心
<400> 3
Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val
1 5 10 15
Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val
20 25 30
Ile Arg
<210> 4
<211> 112
<212> PRT
<213> 人工序列
<220>
<223> PEt 最大
<400> 4
Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro
1 5 10 15
Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu
20 25 30
Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala
35 40 45
Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu
50 55 60
Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln
65 70 75 80
Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu
85 90 95
Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn
100 105 110
<210> 5
<211> 104
<212> PRT
<213> 人工序列
<220>
<223> RAP1 最小
<400> 5
Ala Glu Phe Glu Glu Pro Arg Val Ile Asp Leu Trp Asp Leu Ala Gln
1 5 10 15
Ser Ala Asn Leu Thr Asp Lys Glu Leu Glu Ala Phe Arg Glu Glu Leu
20 25 30
Lys His Phe Glu Ala Lys Ile Glu Lys His Asn His Tyr Gln Lys Gln
35 40 45
Leu Glu Ile Ala His Glu Lys Leu Arg His Ala Glu Ser Val Gly Asp
50 55 60
Gly Glu Arg Val Ser Arg Ser Arg Glu Lys His Ala Leu Leu Glu Gly
65 70 75 80
Arg Thr Lys Glu Leu Gly Tyr Thr Val Lys Lys His Leu Gln Asp Leu
85 90 95
Ser Gly Arg Ile Ser Arg Ala Arg
100
<210> 6
<211> 153
<212> PRT
<213> 人工序列
<220>
<223> A2M 最小
<400> 6
Val Tyr Leu Gln Thr Ser Leu Lys Tyr Asn Ile Leu Pro Glu Lys Glu
1 5 10 15
Glu Phe Pro Phe Ala Leu Gly Val Gln Thr Leu Pro Gln Thr Cys Asp
20 25 30
Glu Pro Lys Ala His Thr Ser Phe Gln Ile Ser Leu Ser Val Ser Tyr
35 40 45
Thr Gly Ser Arg Ser Ala Ser Asn Met Ala Ile Val Asp Val Lys Met
50 55 60
Val Ser Gly Phe Ile Pro Leu Lys Pro Thr Val Lys Met Leu Glu Arg
65 70 75 80
Ser Asn His Val Ser Arg Thr Glu Val Ser Ser Asn His Val Leu Ile
85 90 95
Tyr Leu Asp Lys Val Ser Asn Gln Thr Leu Ser Leu Phe Phe Thr Val
100 105 110
Leu Gln Asp Val Pro Val Arg Asp Leu Lys Pro Ala Ile Val Lys Val
115 120 125
Tyr Asp Tyr Tyr Glu Thr Asp Glu Phe Ala Ile Ala Glu Tyr Asn Ala
130 135 140
Pro Cys Ser Lys Asp Leu Gly Asn Ala
145 150
<210> 7
<211> 24
<212> PRT
<213> 人工序列
<220>
<223> HIV-Tat 最小
<400> 7
Arg Gly Asp Pro Thr Gly Gln Glu Glu Ser Lys Glu Lys Val Glu Lys
1 5 10 15
Glu Thr Val Val Asp Pro Val Thr
20
<210> 8
<211> 641
<212> PRT
<213> 人工序列
<220>
<223> HSPs 最小
<400> 8
Met Ala Lys Ala Ala Ala Ile Gly Ile Asp Leu Gly Thr Thr Tyr Ser
1 5 10 15
Cys Val Gly Val Phe Gln His Gly Lys Val Glu Ile Ile Ala Asn Asp
20 25 30
Gln Gly Asn Arg Thr Thr Pro Ser Tyr Val Ala Phe Thr Asp Thr Glu
35 40 45
Arg Leu Ile Gly Asp Ala Ala Lys Asn Gln Val Ala Leu Asn Pro Gln
50 55 60
Asn Thr Val Phe Asp Ala Lys Arg Leu Ile Gly Arg Lys Phe Gly Asp
65 70 75 80
Pro Val Val Gln Ser Asp Met Lys His Trp Pro Phe Gln Val Ile Asn
85 90 95
Asp Gly Asp Lys Pro Lys Val Gln Val Ser Tyr Lys Gly Glu Thr Lys
100 105 110
Ala Phe Tyr Pro Glu Glu Ile Ser Ser Met Val Leu Thr Lys Met Lys
115 120 125
Glu Ile Ala Glu Ala Tyr Leu Gly Tyr Pro Val Thr Asn Ala Val Ile
130 135 140
Thr Val Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys Asp
145 150 155 160
Ala Gly Val Ile Ala Gly Leu Asn Val Leu Arg Ile Ile Asn Glu Pro
165 170 175
Thr Ala Ala Ala Ile Ala Tyr Gly Leu Asp Arg Thr Gly Lys Gly Glu
180 185 190
Arg Asn Val Leu Ile Phe Asp Leu Gly Gly Gly Thr Phe Asp Val Ser
195 200 205
Ile Leu Thr Ile Asp Asp Gly Ile Phe Glu Val Lys Ala Thr Ala Gly
210 215 220
Asp Thr His Leu Gly Gly Glu Asp Phe Asp Asn Arg Leu Val Asn His
225 230 235 240
Phe Val Glu Glu Phe Lys Arg Lys His Lys Lys Asp Ile Ser Gln Asn
245 250 255
Lys Arg Ala Val Arg Arg Leu Arg Thr Ala Cys Glu Arg Ala Lys Arg
260 265 270
Thr Leu Ser Ser Ser Thr Gln Ala Ser Leu Glu Ile Asp Ser Leu Phe
275 280 285
Glu Gly Ile Asp Phe Tyr Thr Ser Ile Thr Arg Ala Arg Phe Glu Glu
290 295 300
Leu Cys Ser Asp Leu Phe Arg Ser Thr Leu Glu Pro Val Glu Lys Ala
305 310 315 320
Leu Arg Asp Ala Lys Leu Asp Lys Ala Gln Ile His Asp Leu Val Leu
325 330 335
Val Gly Gly Ser Thr Arg Ile Pro Lys Val Gln Lys Leu Leu Gln Asp
340 345 350
Phe Phe Asn Gly Arg Asp Leu Asn Lys Ser Ile Asn Pro Asp Glu Ala
355 360 365
Val Ala Tyr Gly Ala Ala Val Gln Ala Ala Ile Leu Met Gly Asp Lys
370 375 380
Ser Glu Asn Val Gln Asp Leu Leu Leu Leu Asp Val Ala Pro Leu Ser
385 390 395 400
Leu Gly Leu Glu Thr Ala Gly Gly Val Met Thr Ala Leu Ile Lys Arg
405 410 415
Asn Ser Thr Ile Pro Thr Lys Gln Thr Gln Ile Phe Thr Thr Tyr Ser
420 425 430
Asp Asn Gln Pro Gly Val Leu Ile Gln Val Tyr Glu Gly Glu Arg Ala
435 440 445
Met Thr Lys Asp Asn Asn Leu Leu Gly Arg Phe Glu Leu Ser Gly Ile
450 455 460
Pro Pro Ala Pro Arg Gly Val Pro Gln Ile Glu Val Thr Phe Asp Ile
465 470 475 480
Asp Ala Asn Gly Ile Leu Asn Val Thr Ala Thr Asp Lys Ser Thr Gly
485 490 495
Lys Ala Asn Lys Ile Thr Ile Thr Asn Asp Lys Gly Arg Leu Ser Lys
500 505 510
Glu Glu Ile Glu Arg Met Val Gln Glu Ala Glu Lys Tyr Lys Ala Glu
515 520 525
Asp Glu Val Gln Arg Glu Arg Val Ser Ala Lys Asn Ala Leu Glu Ser
530 535 540
Tyr Ala Phe Asn Met Lys Ser Ala Val Glu Asp Glu Gly Leu Lys Gly
545 550 555 560
Lys Ile Ser Glu Ala Asp Lys Lys Lys Val Leu Asp Lys Cys Gln Glu
565 570 575
Val Ile Ser Trp Leu Asp Ala Asn Thr Leu Ala Glu Lys Asp Glu Phe
580 585 590
Glu His Lys Arg Lys Glu Leu Glu Gln Val Cys Asn Pro Ile Ile Ser
595 600 605
Gly Leu Tyr Gln Gly Ala Gly Gly Pro Gly Pro Gly Gly Phe Gly Ala
610 615 620
Gln Gly Pro Lys Gly Gly Ser Gly Ser Gly Pro Thr Ile Glu Glu Val
625 630 635 640
Asp
<210> 9
<211> 252
<212> PRT
<213> 假单胞菌
<400> 9
Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val
1 5 10 15
Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro
20 25 30
Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val
35 40 45
Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu
50 55 60
Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu
65 70 75 80
Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser
85 90 95
Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn
100 105 110
Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His
115 120 125
Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys
130 135 140
Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu
145 150 155 160
Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met
165 170 175
Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser
180 185 190
Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Tyr
195 200 205
Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys Ile
210 215 220
Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His Asp Leu Asp Ile Lys
225 230 235 240
Pro Thr Val Ile Ser His Arg Leu His Phe Pro Glu
245 250
<210> 10
<211> 613
<212> PRT
<213> 假单胞菌
<400> 10
Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val
1 5 10 15
Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro
20 25 30
Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val
35 40 45
Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu
50 55 60
Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu
65 70 75 80
Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser
85 90 95
Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn
100 105 110
Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His
115 120 125
Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys
130 135 140
Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu
145 150 155 160
Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met
165 170 175
Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser
180 185 190
Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Tyr
195 200 205
Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys Ile
210 215 220
Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His Asp Leu Asp Ile Lys
225 230 235 240
Pro Thr Val Ile Ser His Arg Leu His Phe Pro Glu Gly Gly Ser Leu
245 250 255
Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe
260 265 270
Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly
275 280 285
Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser
290 295 300
Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly
305 310 315 320
Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala
325 330 335
Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg
340 345 350
Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val
355 360 365
Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp
370 375 380
Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe
385 390 395 400
Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn
405 410 415
Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg
420 425 430
Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln
435 440 445
Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala
450 455 460
Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly
465 470 475 480
Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly
485 490 495
Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr
500 505 510
Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu
515 520 525
Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly
530 535 540
Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu
545 550 555 560
Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg
565 570 575
Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln
580 585 590
Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro
595 600 605
Arg Glu Asp Leu Lys
610
<210> 11
<211> 323
<212> PRT
<213> 人类
<400> 11
Tyr Ser Arg Glu Lys Asn Gln Pro Lys Pro Ser Pro Lys Arg Glu Ser
1 5 10 15
Gly Glu Glu Phe Arg Met Glu Lys Leu Asn Gln Leu Trp Glu Lys Ala
20 25 30
Gln Arg Leu His Leu Pro Pro Val Arg Leu Ala Glu Leu His Ala Asp
35 40 45
Leu Lys Ile Gln Glu Arg Asp Glu Leu Ala Trp Lys Lys Leu Lys Leu
50 55 60
Asp Gly Leu Asp Glu Asp Gly Glu Lys Glu Ala Arg Leu Ile Arg Asn
65 70 75 80
Leu Asn Val Ile Leu Ala Lys Tyr Gly Leu Asp Gly Lys Lys Asp Ala
85 90 95
Arg Gln Val Thr Ser Asn Ser Leu Ser Gly Thr Gln Glu Asp Gly Leu
100 105 110
Asp Asp Pro Arg Leu Glu Lys Leu Trp His Lys Ala Lys Thr Ser Gly
115 120 125
Lys Phe Ser Gly Glu Glu Leu Asp Lys Leu Trp Arg Glu Phe Leu His
130 135 140
His Lys Glu Lys Val His Glu Tyr Asn Val Leu Leu Glu Thr Leu Ser
145 150 155 160
Arg Thr Glu Glu Ile His Glu Asn Val Ile Ser Pro Ser Asp Leu Ser
165 170 175
Asp Ile Lys Gly Ser Val Leu His Ser Arg His Thr Glu Leu Lys Glu
180 185 190
Lys Leu Arg Ser Ile Asn Gln Gly Leu Asp Arg Leu Arg Arg Val Ser
195 200 205
His Gln Gly Tyr Ser Thr Glu Ala Glu Phe Glu Glu Pro Arg Val Ile
210 215 220
Asp Leu Trp Asp Leu Ala Gln Ser Ala Asn Leu Thr Asp Lys Glu Leu
225 230 235 240
Glu Ala Phe Arg Glu Glu Leu Lys His Phe Glu Ala Lys Ile Glu Lys
245 250 255
His Asn His Tyr Gln Lys Gln Leu Glu Ile Ala His Glu Lys Leu Arg
260 265 270
His Ala Glu Ser Val Gly Asp Gly Glu Arg Val Ser Arg Ser Arg Glu
275 280 285
Lys His Ala Leu Leu Glu Gly Arg Thr Lys Glu Leu Gly Tyr Thr Val
290 295 300
Lys Lys His Leu Gln Asp Leu Ser Gly Arg Ile Ser Arg Ala Arg His
305 310 315 320
Asn Glu Leu
<210> 12
<211> 357
<212> PRT
<213> 人类
<400> 12
Met Ala Pro Arg Arg Val Arg Ser Phe Leu Arg Gly Leu Pro Ala Leu
1 5 10 15
Leu Leu Leu Leu Leu Phe Leu Gly Pro Trp Pro Ala Ala Ser His Gly
20 25 30
Gly Lys Tyr Ser Arg Glu Lys Asn Gln Pro Lys Pro Ser Pro Lys Arg
35 40 45
Glu Ser Gly Glu Glu Phe Arg Met Glu Lys Leu Asn Gln Leu Trp Glu
50 55 60
Lys Ala Gln Arg Leu His Leu Pro Pro Val Arg Leu Ala Glu Leu His
65 70 75 80
Ala Asp Leu Lys Ile Gln Glu Arg Asp Glu Leu Ala Trp Lys Lys Leu
85 90 95
Lys Leu Asp Gly Leu Asp Glu Asp Gly Glu Lys Glu Ala Arg Leu Ile
100 105 110
Arg Asn Leu Asn Val Ile Leu Ala Lys Tyr Gly Leu Asp Gly Lys Lys
115 120 125
Asp Ala Arg Gln Val Thr Ser Asn Ser Leu Ser Gly Thr Gln Glu Asp
130 135 140
Gly Leu Asp Asp Pro Arg Leu Glu Lys Leu Trp His Lys Ala Lys Thr
145 150 155 160
Ser Gly Lys Phe Ser Gly Glu Glu Leu Asp Lys Leu Trp Arg Glu Phe
165 170 175
Leu His His Lys Glu Lys Val His Glu Tyr Asn Val Leu Leu Glu Thr
180 185 190
Leu Ser Arg Thr Glu Glu Ile His Glu Asn Val Ile Ser Pro Ser Asp
195 200 205
Leu Ser Asp Ile Lys Gly Ser Val Leu His Ser Arg His Thr Glu Leu
210 215 220
Lys Glu Lys Leu Arg Ser Ile Asn Gln Gly Leu Asp Arg Leu Arg Arg
225 230 235 240
Val Ser His Gln Gly Tyr Ser Thr Glu Ala Glu Phe Glu Glu Pro Arg
245 250 255
Val Ile Asp Leu Trp Asp Leu Ala Gln Ser Ala Asn Leu Thr Asp Lys
260 265 270
Glu Leu Glu Ala Phe Arg Glu Glu Leu Lys His Phe Glu Ala Lys Ile
275 280 285
Glu Lys His Asn His Tyr Gln Lys Gln Leu Glu Ile Ala His Glu Lys
290 295 300
Leu Arg His Ala Glu Ser Val Gly Asp Gly Glu Arg Val Ser Arg Ser
305 310 315 320
Arg Glu Lys His Ala Leu Leu Glu Gly Arg Thr Lys Glu Leu Gly Tyr
325 330 335
Thr Val Lys Lys His Leu Gln Asp Leu Ser Gly Arg Ile Ser Arg Ala
340 345 350
Arg His Asn Glu Leu
355
<210> 13
<211> 101
<212> PRT
<213> 人免疫缺陷病毒
<400> 13
Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Pro Cys Thr Lys Cys Tyr Cys Lys Lys Cys Cys Leu
20 25 30
His Cys Gln Val Cys Phe Met Thr Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Asn Lys Asn
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Thr Ser Arg Ala Arg Gly Asp
65 70 75 80
Pro Thr Gly Gln Glu Glu Ser Lys Glu Lys Val Glu Lys Glu Thr Val
85 90 95
Val Asp Pro Val Thr
100
<210> 14
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> ER 滞留序列
<400> 14
Lys Asp Glu Leu
1
<210> 15
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> CD28-PEt的连接子
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa可为任一种天然氨基酸
<220>
<221> misc_feature
<222> (4)..(4)
<223> Xaa可为任一种天然氨基酸
<400> 15
Arg Xaa Arg Xaa Lys Arg
1 5
<210> 16
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> ER 滞留序列
<400> 16
Lys Lys Asp Leu Arg Asp Glu Leu Lys Asp Glu Leu
1 5 10
<210> 17
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> ER 滞留序列
<400> 17
Lys Lys Asp Glu Leu Arg Asp Glu Leu Lys Asp Glu Leu
1 5 10
<210> 18
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> ER 滞留序列
<400> 18
Lys Lys Asp Glu Leu Arg Val Glu Leu Lys Asp Glu Leu
1 5 10
<210> 19
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> ER 滞留序列
<400> 19
Lys Asp Glu Leu Lys Asp Glu Leu Lys Asp Glu Leu
1 5 10
<210> 20
<211> 46
<212> PRT
<213> 假单胞菌
<400> 20
Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln
1 5 10 15
Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu
20 25 30
Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg
35 40 45
<210> 21
<211> 98
<212> PRT
<213> 人乳头瘤病毒第16型
<400> 21
Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln
1 5 10 15
Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser
20 25 30
Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp
35 40 45
Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr
50 55 60
Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu
65 70 75 80
Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln
85 90 95
Lys Pro
<210> 22
<211> 105
<212> PRT
<213> 人乳头瘤病毒第18型
<400> 22
Met His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu Glu
1 5 10 15
Pro Gln Asn Glu Ile Pro Val Asp Leu Leu Cys His Glu Gln Leu Ser
20 25 30
Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly Val Asn His Gln His
35 40 45
Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met Leu Cys Met
50 55 60
Cys Cys Lys Cys Glu Ala Arg Ile Lys Leu Val Val Glu Ser Ser Ala
65 70 75 80
Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr Leu Ser Phe
85 90 95
Val Cys Pro Trp Cys Ala Ser Gln Gln
100 105
<210> 23
<211> 190
<212> PRT
<213> 丙型肝炎病毒
<400> 23
Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn
1 5 10 15
Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly
20 25 30
Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala
35 40 45
Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro
50 55 60
Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly
65 70 75 80
Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Met Gly Trp Ala Gly Trp
85 90 95
Leu Leu Ser Pro Arg Gly Ser Arg Pro Asn Trp Gly Pro Thr Asp Pro
100 105 110
Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys
115 120 125
Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu
130 135 140
Gly Gly Val Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp
145 150 155 160
Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile
165 170 175
Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Thr Pro Ala Ser
180 185 190
<210> 24
<211> 154
<212> PRT
<213> 乙型肝炎病毒
<400> 24
Met Ala Ala Arg Met Cys Cys Gln Leu Asp Pro Ala Arg Asp Val Leu
1 5 10 15
Cys Leu Arg Pro Val Gly Ala Glu Ser Arg Gly Arg Pro Leu Pro Gly
20 25 30
Pro Leu Gly Ala Leu Pro Pro Ser Ser Ala Ser Ala Val Pro Ala Asp
35 40 45
His Gly Ser His Leu Ser Leu Arg Gly Leu Pro Val Cys Ser Phe Ser
50 55 60
Ser Ala Gly Pro Cys Ala Leu Arg Phe Thr Ser Ala Arg Arg Met Glu
65 70 75 80
Thr Thr Val Asn Ala Pro Trp Ser Leu Pro Thr Val Leu His Lys Arg
85 90 95
Thr Ile Gly Leu Ser Gly Arg Ser Met Thr Trp Ile Glu Glu Tyr Ile
100 105 110
Lys Asp Cys Val Phe Lys Asp Trp Glu Glu Leu Gly Glu Glu Ile Arg
115 120 125
Leu Lys Val Phe Val Leu Gly Gly Cys Arg His Lys Leu Val Cys Ser
130 135 140
Pro Ala Pro Cys Asn Phe Phe Thr Ser Ala
145 150
<210> 25
<211> 192
<212> PRT
<213> 猪环状病毒
<400> 25
Asn Gly Ile Phe Asn Thr Arg Leu Ser Arg Thr Phe Gly Tyr Thr Ile
1 5 10 15
Lys Arg Thr Thr Val Lys Thr Pro Ser Trp Ala Val Asp Met Met Arg
20 25 30
Phe Asn Ile Asn Asp Phe Leu Pro Pro Gly Gly Gly Ser Asn Pro Arg
35 40 45
Ser Val Pro Phe Glu Tyr Tyr Ser Ile Ser Lys Val Lys Val Glu Phe
50 55 60
Trp Pro Cys Ser Pro Ile Thr Gln Gly Asp Ser Gly Val Gly Ser Ser
65 70 75 80
Ala Val Ile Leu Asp Asp Asn Phe Val Thr Lys Ala Thr Ala Leu Thr
85 90 95
Tyr Asp Pro Tyr Val Asn Tyr Ser Ser Arg His Thr Ile Thr Gln Pro
100 105 110
Phe Ser Tyr His Ser Arg Tyr Phe Thr Pro Lys Pro Val Leu Asp Ser
115 120 125
Thr Ile Asp Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp Leu
130 135 140
Arg Leu Gln Thr Ala Gly Asn Val Asp His Val Gly Leu Gly Thr Ala
145 150 155 160
Phe Glu Asn Ser Ile Tyr Asp Gln Glu Tyr Asn Ile Arg Val Thr Met
165 170 175
Tyr Val Gln Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu Asn Pro
180 185 190
<210> 26
<211> 220
<212> PRT
<213> 猪繁殖与呼吸综合症病毒
<400> 26
Arg His His Phe Thr Pro Ser Glu Arg Gln Leu Cys Leu Ser Ser Ile
1 5 10 15
Gln Thr Ala Phe Asn Gln Gly Ala Gly Thr Cys Ile Leu Ser Asp Ser
20 25 30
Gly Arg Ile Ser Tyr Thr Val Glu Phe Ser Leu Pro Thr His His Thr
35 40 45
Val Arg Leu Ile Arg Val Thr Ala Pro Pro Ser Ala Leu Asp Gln Val
50 55 60
Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu
65 70 75 80
Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala
85 90 95
Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu
100 105 110
Ala Gly Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala
115 120 125
Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu
130 135 140
Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val
145 150 155 160
Arg His His Phe Thr Pro Ser Glu Arg Gln Leu Cys Leu Ser Ser Ile
165 170 175
Gln Thr Ala Phe Asn Gln Gly Ala Gly Thr Cys Ile Leu Ser Asp Ser
180 185 190
Gly Arg Ile Ser Tyr Thr Val Glu Phe Ser Leu Pro Thr His His Thr
195 200 205
Val Arg Leu Ile Arg Val Thr Ala Pro Pro Ser Ala
210 215 220
<210> 27
<211> 165
<212> PRT
<213> 猪繁殖与呼吸综合症病毒
<400> 27
Asn Asn Lys Glu Cys Thr Val Ala Gln Ala Leu Gly Asn Gly Asp Lys
1 5 10 15
Phe Arg Ala Thr Asp Lys Arg Val Val Asp Ser Leu Arg Ala Ile Cys
20 25 30
Ala Asp Leu Glu Gly Ser Ser Ser Pro Leu Pro Lys Val Ala His Asn
35 40 45
Leu Gly Phe Tyr Phe Ser Pro Asp Leu Thr Gln Phe Ala Lys Leu Pro
50 55 60
Ile Glu Leu Asp Pro His Trp Pro Val Val Ser Thr Gln Asn Asn Glu
65 70 75 80
Lys Trp Pro Asp Arg Leu Val Ala Ser Leu Arg Pro Leu Asp Lys Tyr
85 90 95
Ser Arg Ala Cys Ile Gly Ala Gly Tyr Met Val Gly Pro Ser Val Phe
100 105 110
Leu Gly Thr Pro Gly Val Val Ser Tyr Tyr Leu Thr Lys Phe Val Lys
115 120 125
Gly Glu Ala Gln Val Leu Pro Glu Thr Val Phe Ser Thr Gly Arg Ile
130 135 140
Glu Val Asp Cys Arg Glu Tyr Leu Asp Asp Arg Glu Arg Glu Val Ala
145 150 155 160
Ala Ser Leu Pro His
165
<210> 28
<211> 58
<212> PRT
<213> 猪繁殖与呼吸综合症病毒
<400> 28
Gly Ser Ser Leu Asp Asp Phe Cys Tyr Asp Ser Thr Ala Pro Gln Lys
1 5 10 15
Val Leu Leu Ala Phe Ser Ile Thr Tyr Ala Ser Asn Asp Ser Ser Ser
20 25 30
His Leu Gln Leu Ile Tyr Asn Leu Thr Leu Cys Glu Leu Asn Gly Thr
35 40 45
Asp Trp Leu Ala Asn Lys Phe Asp Trp Ala
50 55
<210> 29
<211> 62
<212> PRT
<213> 猪繁殖与呼吸综合症病毒
<400> 29
Met Gly Ser Leu Asp Asp Phe Cys Asn Asp Ser Thr Ala Ala Gln Lys
1 5 10 15
Leu Val Leu Ala Phe Ser Ile Thr Tyr Thr Pro Ile Phe Val Ala Gly
20 25 30
Gly Ser Ser Ser Thr Tyr Gln Tyr Ile Tyr Asn Leu Thr Ile Cys Glu
35 40 45
Leu Asn Gly Thr Asp Trp Leu Ser Asn His Phe Asp Trp Ala
50 55 60
<210> 30
<211> 68
<212> PRT
<213> 人工序列
<220>
<223> CD28-PEt
<220>
<221> misc_feature
<222> (8)..(8)
<223> Xaa可为任一种天然氨基酸
<220>
<221> misc_feature
<222> (10)..(11)
<223> Xaa可为任一种天然氨基酸
<220>
<221> misc_feature
<222> (17)..(17)
<223> Xaa可为任一种天然氨基酸
<220>
<221> misc_feature
<222> (30)..(30)
<223> Xaa可为任一种天然氨基酸
<220>
<221> misc_feature
<222> (32)..(32)
<223> Xaa可为任一种天然氨基酸
<400> 30
Thr Asp Ile Tyr Phe Cys Lys Xaa Glu Xaa Xaa Tyr Pro Pro Pro Tyr
1 5 10 15
Xaa Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His Arg Xaa Arg Xaa
20 25 30
Lys Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg
35 40 45
Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp
50 55 60
Gln Val Ile Arg
65
<210> 31
<211> 28
<212> PRT
<213> 人工序列
<220>
<223> CD28共有序列
<220>
<221> misc_feature
<222> (8)..(8)
<223> Xaa可为任一种天然氨基酸
<220>
<221> misc_feature
<222> (10)..(11)
<223> Xaa可为任一种天然氨基酸
<220>
<221> misc_feature
<222> (17)..(17)
<223> Xaa可为任一种天然氨基酸
<400> 31
Thr Asp Ile Tyr Phe Cys Lys Xaa Glu Xaa Xaa Tyr Pro Pro Pro Tyr
1 5 10 15
Xaa Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His
20 25
<210> 32
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> CD28关键区
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa可为任一种天然氨基酸
<220>
<221> misc_feature
<222> (4)..(5)
<223> Xaa可为任一种天然氨基酸
<400> 32
Lys Xaa Glu Xaa Xaa Tyr Pro Pro Pro Tyr
1 5 10

Claims (14)

1.一种融合蛋白，其包含：
(a)抗原呈递细胞(APC)结合域或CD91受体结合域，其位于所述融合蛋白的N端，所述APC结合域或CD91受体结合域选自受体相关蛋白-1(RAP1)区域III、α-2-巨球蛋白受体相关蛋白(A2M)、HIV-Tat、热休克蛋白(HSP)及假单胞菌外毒素A(PE)结合域I；
(b)蛋白转导域，其位于所述APC结合域或CD91受体结合域的C端，所述蛋白转导域选自由下述(i)及(ii)所组成的群组：
(i)融合多肽，其由T细胞致敏信号转导肽、连接子及PE转位肽所组成，其中：
(1)所述T细胞致敏信号转导肽位于所述融合多肽的N端，其由SEQ ID NO:31、SEQ IDNO:1或SEQ ID NO:2的氨基酸序列所组成，其中，所述SEQ ID NO:31的Xaa8为I或L；Xaa10为V、F或A；Xaa11为M或L；Xaa17为L或I；
(2)所述连接子由SEQ ID NO:15的氨基酸序列所组成，其连接所述T细胞致敏信号转导肽与所述PE转位肽；且
(3)所述PE转位肽为选自SEQ ID NO:3、SEQ ID NO:20或SEQID NO:4的氨基酸序列；及
(ii)T细胞致敏信号转导肽，其由SEQ ID NO:31、SEQ ID NO:1或SEQ ID NO:2的氨基酸序列所组成，其中，所述SEQ ID NO:31的Xaa8为I或L；Xaa10为V、F或A；Xaa11为M或L；Xaa17为L或I；及
(c)病原体的抗原，其位于所述蛋白转导域的C端，所述病原体为乙型肝炎病毒(HBV)。
2.如权利要求1所述的融合蛋白，其中，所述蛋白转导域为所述融合多肽。
3.如权利要求1所述的融合蛋白，其中，所述受体相关蛋白-1(RAP1)区域III、α-2-巨球蛋白受体相关蛋白(A2M)、HIV-Tat、热休克蛋白(HSP)及假单胞菌外毒素A(PE)结合域I分别为由SEQ ID NO:5的氨基酸序列所组成的多肽、由SEQ ID NO:6的氨基酸序列所组成的多肽、由SEQ ID NO:7的氨基酸序列所组成的多肽、由SEQ ID NO:8的氨基酸序列所组成的多肽及由SEQ ID NO:9的氨基酸序列所组成的多肽。
4.如权利要求1所述的融合蛋白，进一步包含位于所述融合蛋白的C端的内质网滞留序列。
5.如权利要求4所述的融合蛋白，其中，所述内质网滞留序列选自由SEQ ID NO:16、SEQID NO:17、SEQ ID NO:18及SEQ ID NO:19所组成的群组的氨基酸序列。
6.如权利要求1所述的融合蛋白，其中，所述蛋白转导域由SEQ ID NO:30的氨基酸序列所组成。
7.如权利要求1所述的融合蛋白，其中，所述受体相关蛋白-1(RAP1)区域III以及假单胞菌外毒素A(PE)结合域I分别为由SEQ ID NO:5的氨基酸序列所组成的多肽及由SEQ IDNO:9的氨基酸序列所组成的多肽。
8.如权利要求1所述的融合蛋白，其中，所述T细胞致敏信号转导肽由SEQ ID NO:1或SEQ ID NO:2的氨基酸序列所组成。
9.一种融合蛋白，其包含：
(a)抗原呈递细胞(APC)结合域，其位于所述融合蛋白的N端，其中所述APC结合域为选自SEQ ID NO:5、SEQ ID NO:7或SEQ ID NO:8的氨基酸序列；
(b)蛋白转导域，其位于所述APC结合域的C端，所述蛋白转导域为长度为34-46个氨基酸残基的假单胞菌外毒素A(PE)转位肽，其选自SEQ ID NO:3或SEQ ID NO:20的氨基酸序列；
(c)病原体的抗原，其位于所述蛋白转导域的C端，所述病原体为乙型肝炎病毒(HBV)；及
(d)内质网滞留序列，其位于所述融合蛋白的C端。
10.如权利要求9所述的融合蛋白，其在所述APC结合域与所述PE转位肽之间进一步包含另一连接子，所述另一连接子由SEQ ID NO:15的氨基酸序列所组成。
11.如权利要求1或9所述的融合蛋白，其中，所述病原体的抗原为乙型肝炎病毒(HBV)X蛋白。
12.如权利要求1或9所述的融合蛋白，其中，所述病原体的抗原为由SEQ ID NO:24所组成的氨基酸序列。
13.一种如权利要求1-12中任一项所述的融合蛋白在制造用于诱导增强的病原体抗原特异性T细胞反应的药物中的用途。
14.一种如权利要求1-12中任一项所述的融合蛋白在制造用于预防、治疗由病原体所引起的感染和/或将由感染所引起的症状减至最少的药物中的用途。
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